The Ighmbp2D564N mouse model is the first SMARD1 model to demonstrate respiratory defects.

Spinal muscular atrophy with respiratory distress type I (SMARD1) is a neurodegenerative disease defined by respiratory distress, muscle atrophy and sensory and autonomic nervous system defects. SMARD1 is a result of mutations within the IGHMBP2 gene. We have generated six Ighmbp2 mouse models based on patient-derived mutations that result in SMARD1 and/or Charcot-Marie Tooth Type 2 (CMT2S). Here we describe the characterization of one of these models, Ighmbp2D564N (human D565N). The Ighmbp2D564N/D564N mouse model mimics important aspects of the SMARD1 disease phenotype, including motor neuron degeneration and muscle atrophy. Ighmbp2D564N/D564N is the first SMARD1 mouse model to demonstrate respiratory defects based on quantified plethysmography analyses. SMARD1 disease phenotypes, including the respiratory defects, are significantly diminished by intracerebroventricular (ICV) injection of ssAAV9-IGHMBP2 and the extent of phenotypic restoration is dose-dependent. Collectively, this model provides important biological insight into SMARD1 disease development.

Impact of Limb Phenotype on Tongue Denervation Atrophy, Dysphagia Penetrance, and Survival Time in a Mouse Model of ALS.

Current treatments for dysphagia in ALS do not target the underlying tongue weakness and denervation atrophy that is prevalent in spinal and bulbar ALS cases. To address this clinical gap, we studied the low copy number SOD1-G93A (LCN-SOD1) mouse model of ALS to quantify the impact of limb phenotype on tongue denervation atrophy, dysphagia penetrance, and survival time in preparation for future treatment-based studies. Two male LCN-SOD1 breeders and 125 offspring were followed for limb phenotype inheritance, of which 52 (30 LCN-SOD1 and 22 wild-type/WT, both sexes) underwent characterization of dysphagia penetrance (via videofluoroscopic swallow study; VFSS) and survival time at disease end-stage (15-20% body weight loss). From these, 16 mice (8/genotype) underwent postmortem histological analysis of the genioglossus for evidence of denervation atrophy. Results revealed that both breeders displayed a mixed (hindlimb and forelimb) ALS phenotype and sired equal proportions of hindlimb vs. mixed phenotype offspring. Dysphagia penetrance was complete for mixed (100%) versus incomplete for hindlimb (64%) phenotype mice; yet survival times were similar. Regardless of limb phenotype, LCN-SOD1 mice had significantly smaller genioglossus myofibers and more centralized myonuclei compared to WT mice (p < 0.05). These biomarkers of denervation atrophy were significantly correlated with VFSS metrics (lick and swallow rates, p < 0.05) but not survival time. In conclusion, both LCN-SOD1 phenotypes had significant tongue denervation atrophy, even hindlimb phenotype mice without dysphagia. This finding recapitulates human ALS, providing robust rationale for using this preclinical model to explore targeted treatments for tongue denervation atrophy and ensuing dysphagia.

Mechanisms of severe acute intermittent hypoxia-induced phrenic long-term facilitation.

Moderate acute intermittent hypoxia (mAIH; 35-55 mmHg PaO2) elicits phrenic long-term facilitation (pLTF) by a mechanism that requires activation of Gq protein-coupled serotonin type 2 receptors, MEK/ERK MAP kinase, and NADPH oxidase activity and is constrained by cAMP-PKA signaling. In contrast, severe AIH (sAIH; 25-35 mmHg PaO2) elicits Gs protein-coupled adenosine type 2 A receptor-dependent pLTF. Another Gs protein-coupled receptor, serotonin 7 receptors, elicits phrenic motor facilitation (pMF) by a mechanism that requires exchange protein activated by cyclic AMP (EPAC) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and is constrained by NADPH oxidase activity. Here, we tested the hypothesis that the same downstream signaling mechanisms giving rise to serotonin 7 (vs. serotonin 2) receptor-induced pMF underlie sAIH-induced pLTF. In anesthetized rats, sAIH-induced pLTF was compared after pretreatment with intrathecal (C4) injections of inhibitors for: 1) EPAC (ESI-05); 2) MEK/ERK (UO126); 3) PKA (KT-5720); 4) PI3K/Akt (PI828); and 5) NADPH oxidase (apocynin). In partial agreement with our hypothesis, sAIH-induced pLTF was abolished by ESI-05 and PI828 and marginally enhanced by apocynin but, surprisingly, was abolished by UO126 and attenuated by KT-5720. Mechanisms of sAIH-induced pLTF reflect elements of both Gq and Gs pathways to pMF, likely as a consequence of the complex, cross-talk interactions between them.NEW & NOTEWORTHY Distinct mechanisms give rise to pLTF induced by moderate and severe AIH. We demonstrate that, unlike moderate AIH, severe AIH-induced pLTF requires EPAC and PI3K/Akt and is marginally constrained by NADPH oxidase activity. Surprisingly, sAIH-induced pLTF requires MEK/ERK activity similar to moderate AIH-induced pLTF and is reduced by PKA inhibition. We suggest sAIH-induced pLTF arises from complex interactions between dominant mechanisms characteristic of moderate versus severe AIH-induced pLTF.

Divergent receptor utilization is necessary for phrenic long-term facilitation over the course of motor neuron loss following CTB-SAP intrapleural injections.

Intrapleural injection of cholera toxin B conjugated to saporin (CTB-SAP) mimics respiratory motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Seven-day CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) primarily through TrkB and PI3K/Akt-dependent mechanisms [i.e., Gs-pathway, which can be initiated by adenosine 2A (A2A) receptors in naïve rats], whereas 28-day CTB-SAP rats elicit moderate pLTF though BDNF- and MEK-/ERK-dependent mechanisms [i.e., Gq-pathway, which is typically initiated by serotonin (5-HT) receptors in naïve rats]. Here, we tested the hypothesis that pLTF following CTB-SAP is 1) A2A receptor-dependent at 7 days and 2) 5-HT receptor-dependent at 28 days. Adult Sprague-Dawley male rats were anesthetized, paralyzed, ventilated, and exposed to acute intermittent hypoxia (AIH; 3-, 5-min bouts of 10.5% O2) following bilateral, intrapleural injections at 7 days and 28 days of 1) CTB-SAP (25 µg) or 2) unconjugated CTB and SAP (control). Intrathecal C4 delivery included either the 1) A2A receptor antagonist (MSX-3; 10 µM; 12 µL) or 2) 5-HT receptor antagonist (methysergide; 20 mM; 15 µL). pLTF was abolished with A2A receptor inhibition in 7-day, not 28-day, CTB-SAP rats versus controls (P < 0.05), whereas pLTF was abolished following 5-HT receptor inhibition in 28-day, not 7-day, CTB-SAP rats versus controls (P < 0.05). In addition, 5-HT2A receptor expression was unchanged in CTB-SAP rats versus controls, whereas 5-HT2B receptor expression was decreased in CTB-SAP rats versus controls (P < 0.05). This study furthers our understanding of the contribution of differential receptor activation to pLTF and its implications for breathing following respiratory motor neuron death.NEW & NOTEWORTHY The current study investigates underlying receptor-dependent mechanisms contributing to phrenic long-term facilitation (pLTF) following CTB-SAP-induced respiratory motor neuron death at 7 days and 28 days. We found that A2A receptors are required for enhanced pLTF in 7-day CTB-SAP rats, whereas 5-HT receptors are required for moderate pLTF in 28-day CTB-SAP rats. Targeting these time-dependent mechanisms have implications for breathing maintenance over the course of many neuromuscular diseases.

Tongue and hypoglossal morphology after intralingual cholera toxin B-saporin injection.

INTRODUCTION: We recently developed an inducible model of dysphagia using intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) to cause death of hypoglossal neurons. In this study we aimed to evaluate tongue morphology and ultrastructural changes in hypoglossal neurons and nerve fibers in this model. METHODS: Tissues were collected from 20 rats (10 control and 10 CTB-SAP animals) on day 9 post-injection. Tongues were weighed, measured, and analyzed for microscopic changes using laminin immunohistochemistry. Hypoglossal neurons and axons were examined using transmission electron microscopy. RESULTS: The cross-sectional area of myofibers in the posterior genioglossus was decreased in CTB-SAP-injected rats. Degenerative changes were observed in both the cell bodies and distal axons of hypoglossal neurons. DISCUSSION: Preliminary results indicate this model may have translational application to a variety of neurodegenerative diseases resulting in tongue dysfunction and associated dysphagia.

Peripheral-to-central immune communication at the area postrema glial-barrier following bleomycin-induced sterile lung injury in adult rats.

The pathways for peripheral-to-central immune communication (P â†’ C I-comm) following sterile lung injury (SLI) are unknown. SLI evokes systemic and central inflammation, which alters central respiratory control and viscerosensory transmission in the nucleus tractus solitarii (nTS). These functional changes coincide with increased interleukin-1 beta (IL-1ß) in the area postrema, a sensory circumventricular organ that connects P â†’ C I-comm to brainstem circuits that control homeostasis. We hypothesize that IL-1ß and its downstream transcriptional target, cyclooxygenase-2 (COX-2), mediate P â†’ C I-comm in the nTS. In a rodent model of SLI induced by intratracheal bleomycin (Bleo), the sigh frequency and duration of post-sigh apnea increased in Bleo- compared to saline- treated rats one week after injury. This SLI-dependent change in respiratory control occurred concurrently with augmented IL-1ß and COX-2 immunoreactivity (IR) in the funiculus separans (FS), a barrier between the AP and the brainstem. At this barrier, increases in IL-1ß and COX-2 IR were confined to processes that stained for glial fibrillary acidic protein (GFAP) and that projected basolaterally to the nTS. Further, FS radial-glia did not express TNF-α or IL-6 following SLI. To test our hypothesis, we blocked central COX-1/2 activity by intracerebroventricular (ICV) infusion of Indomethacin (Ind). Continuous ICV Ind treatment prevented Bleo-dependent increases in GFAP + and IL-1ß + IR, and restored characteristics of sighs that reset the rhythm. These data indicate that changes in sighs following SLI depend partially on activation of a central COX-dependent P â†’ C I-comm via radial-glia of the FS.

Optimizing the Translational Value of Mouse Models of ALS for Dysphagia Therapeutic Discovery.

The goal of this study was to compare dysphagia phenotypes in low and high copy number (LCN and HCN) transgenic superoxide dismutase 1 (SOD1) mouse models of ALS to accelerate the discovery of novel and effective treatments for dysphagia and early amyotrophic lateral sclerosis (ALS) diagnosis. Clinicopathological features of dysphagia were characterized in individual transgenic mice and age-matched controls utilizing videofluoroscopy in conjunction with postmortem assays of the tongue and hypoglossal nucleus. Quantitative PCR accurately differentiated HCN-SOD1 and LCN-SOD1 mice and nontransgenic controls. All HCN-SOD1 mice developed stereotypical paralysis in both hindlimbs. In contrast, LCN-SOD1 mice displayed wide variability in fore- and hindlimb involvement. Lick rate, swallow rate, inter-swallow interval, and pharyngeal transit time were significantly altered in both HCN-SOD1 and LCN-SOD1 mice compared to controls. Tongue weight, tongue dorsum surface area, total tongue length, and caudal tongue length were significantly reduced only in the LCN-SOD1 mice compared to age-matched controls. LCN-SOD1 mice with lower body weights had smaller/lighter weight tongues, and those with forelimb paralysis and slower lick rates died at a younger age. LCN-SOD1 mice had a 32% loss of hypoglossal neurons, which differed significantly when compared to age-matched control mice. These novel findings for LCN-SOD1 mice are congruent with reported dysphagia and associated tongue atrophy and hypoglossal nucleus pathology in human ALS patients, thus highlighting the translational potential of this mouse model in ALS research.

Mechanisms of Enhanced Phrenic Long-Term Facilitation in SOD1G93A Rats.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease, causing muscle paralysis and death from respiratory failure. Effective means to preserve/restore ventilation are necessary to increase the quality and duration of life in ALS patients. At disease end-stage in a rat ALS model (SOD1G93A ), acute intermittent hypoxia (AIH) restores phrenic nerve activity to normal levels via enhanced phrenic long-term facilitation (pLTF). Mechanisms enhancing pLTF in end-stage SOD1G93A rats are not known. Moderate AIH-induced pLTF is normally elicited via cellular mechanisms that require the following: Gq-protein-coupled 5-HT2 receptor activation, new BDNF synthesis, and MEK/ERK signaling (the Q pathway). In contrast, severe AIH elicits pLTF via a distinct mechanism that requires the following: Gs-protein-coupled adenosine 2A receptor activation, new TrkB synthesis, and PI3K/Akt signaling (the S pathway). In end-stage male SOD1G93A rats and wild-type littermates, we investigated relative Q versus S pathway contributions to enhanced pLTF via intrathecal (C4) delivery of small interfering RNAs targeting BDNF or TrkB mRNA, and MEK/ERK (U0126) or PI3 kinase/Akt (PI828) inhibitors. In anesthetized, paralyzed and ventilated rats, moderate AIH-induced pLTF was abolished by siBDNF and UO126, but not siTrkB or PI828, demonstrating that enhanced pLTF occurs via the Q pathway. Although phrenic motor neuron numbers were decreased in end-stage SOD1G93A rats (∼30% survival; p < 0.001), BDNF and phosphorylated ERK expression were increased in spared phrenic motor neurons (p < 0.05), consistent with increased Q-pathway contributions to pLTF. Our results increase understanding of respiratory plasticity and its potential to preserve/restore breathing capacity in ALS.SIGNIFICANCE STATEMENT Since neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), end life via respiratory failure, the ability to harness respiratory motor plasticity to improve breathing capacity could increase the quality and duration of life. In a rat ALS model (SOD1G93A ) we previously demonstrated that spinal respiratory motor plasticity elicited by acute intermittent hypoxia is enhanced at disease end-stage, suggesting greater potential to preserve/restore breathing capacity. Here we demonstrate that enhanced intermittent hypoxia-induced phrenic motor plasticity results from amplification of normal cellular mechanisms versus addition/substitution of alternative mechanisms. Greater understanding of mechanisms underlying phrenic motor plasticity in ALS may guide development of new therapies to preserve and/or restore breathing in ALS patients.

Systemic inflammation inhibits serotonin receptor 2-induced phrenic motor facilitation upstream from BDNF/TrkB signaling.

Although systemic inflammation induced by even a low dose of lipopolysaccharide (LPS, 100 µg/kg) impairs respiratory motor plasticity, little is known concerning cellular mechanisms giving rise to this inhibition. Phrenic motor facilitation (pMF) is a form of respiratory motor plasticity elicited by pharmacological agents applied to the cervical spinal cord, or by acute intermittent hypoxia (AIH; 3, 5-min hypoxic episodes); when elicited by AIH, pMF is known as phrenic long-term facilitation (pLTF). AIH consisting of moderate hypoxic episodes (mAIH, arterial Po2 = 35-55 mmHg) elicits pLTF via the Q pathway to pMF, a mechanism that requires spinal serotonin (5HT2) receptor activation and new brain-derived neurotrophic factor (BDNF) protein synthesis. Although mild systemic inflammation attenuates mAIH-induced pLTF via spinal p38 MAP kinase activation, little is known concerning how p38 MAP kinase activity inhibits the Q pathway. Here, we confirmed that 24 h after a low LPS dose (100 µg/kg ip), mAIH-induced pLTF is greatly attenuated. Similarly, pMF elicited by intrathecal cervical injections of 5HT2A (DOI; 100 µM; 3 × 6 µl) or 5HT2B receptor agonists (BW723C86; 100 µM; 3 × 6 µl) is blocked 24 h post-LPS. When pMF was elicited by intrathecal BDNF (100 ng, 12 µl), pMF was actually enhanced 24 h post-LPS. Thus 5HT2A/2B receptor-induced pMF is impaired downstream from 5HT2 receptor activation, but upstream from BDNF/TrkB signaling. Mechanisms whereby LPS augments BDNF-induced pMF are not yet known. NEW & NOTEWORTHY These experiments give novel insights concerning mechanisms whereby systemic inflammation undermines serotonin-dependent, spinal respiratory motor plasticity, yet enhances brain-derived neurotrophic factor (BDNF)/TrkB signaling in phrenic motor neurons. These insights may guide development of new strategies to elicit functional recovery of breathing capacity in patients with respiratory impairment by reducing (or bypassing) the impact of systemic inflammation characteristic of clinical disorders that compromise breathing.

Sustained Hypoxia Elicits Competing Spinal Mechanisms of Phrenic Motor Facilitation.

UNLABELLED: Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal motor plasticity. Competing mechanisms give rise to phrenic motor facilitation (pMF; a general term including pLTF) depending on the severity of hypoxia within episodes. In contrast, moderate acute sustained hypoxia (mASH) does not elicit pMF. By varying the severity of ASH and targeting competing mechanisms of pMF, we sought to illustrate why moderate AIH (mAIH) elicits pMF but mASH does not. Although mAIH elicits serotonin-dependent pLTF, mASH does not; thus, mAIH-induced pLTF is pattern sensitive. In contrast, severe AIH (sAIH) elicits pLTF through adenosine-dependent mechanisms, likely from greater extracellular adenosine accumulation. Because serotonin- and adenosine-dependent pMF interact via cross talk inhibition, we hypothesized that pMF is obscured because the competing mechanisms of pMF are balanced and offsetting during mASH. Here, we demonstrate the following: (1) blocking spinal A2A receptors with MSX-3 reveals mASH-induced pMF; and (2) sASH elicits A2A-dependent pMF. In anesthetized rats pretreated with intrathecal A2A receptor antagonist injections before mASH (PaO2 = 40-54 mmHg) or sASH (PaO2 = 25-36 mmHg), (1) mASH induced a serotonin-dependent pMF and (2) sASH induced an adenosine-dependent pMF, which was enhanced by spinal serotonin receptor inhibition. Thus, competing adenosine- and serotonin-dependent mechanisms contribute differentially to pMF depending on the pattern/severity of hypoxia. Understanding interactions between these mechanisms has clinical relevance as we develop therapies to treat severe neuromuscular disorders that compromise somatic motor behaviors, including breathing. Moreover, these results demonstrate how competing mechanisms of plasticity can give rise to pattern sensitivity in pLTF. SIGNIFICANCE STATEMENT: Intermittent hypoxia elicits pattern-sensitive spinal plasticity and improves motor function after spinal injury or during neuromuscular disease. Specific mechanisms of pattern sensitivity in this form of plasticity are unknown. We provide evidence that competing mechanisms of phrenic motor facilitation mediated by adenosine 2A and serotonin 2 receptors are differentially expressed, depending on the pattern/severity of hypoxia. Understanding how these distinct mechanisms interact during hypoxic exposures differing in severity and duration will help explain interesting properties of plasticity, such as pattern sensitivity, and may help optimize therapies to restore motor function in patients with neuromuscular disorders that compromise movement.

Adenosine-dependent phrenic motor facilitation is inflammation resistant.

Phrenic motor facilitation (pMF), a form of respiratory plasticity, can be elicited by acute intermittent hypoxia (i.e., phrenic long-term facilitation, pLTF) or direct application of drugs to the cervical spinal cord. Moderate acute intermittent hypoxia (mAIH; 3 × 5-min episodes of 35-50 mmHg arterial Po2, 5-min normoxic intervals) induces pLTF by a serotonin-dependent mechanism; mAIH-induced pLTF is abolished by mild systemic inflammation induced by a low dose of lipopolysaccharide (LPS; 100 µg/kg ip). In contrast, severe acute intermittent hypoxia (sAIH; 3 × 5-min episodes of 25-30 mmHg arterial Po2, 5-min normoxic intervals) elicits pLTF by a distinct, adenosine-dependent mechanism. Since it is not known if systemic LPS blocks the mechanism giving rise to sAIH-induced pLTF, we tested the hypothesis that sAIH-induced pLTF and adenosine 2A (A2A) receptor-induced pMF are insensitive to mild systemic inflammation elicited by the same low dose of LPS. In agreement with our hypothesis, neither sAIH-induced pLTF nor cervical intrathecal A2A receptor agonist (CGS-21680; 200 µM, 10 µl × 3)-induced pMF were affected 24 h post-LPS. Pretreatment with intrathecal A2A receptor antagonist injections (MSX-3; 10 µM, 12 µl) blocked sAIH-induced pLTF 24 h post LPS, confirming that pLTF was adenosine dependent. Our results give insights concerning the differential impact of systemic inflammation and the functional significance of multiple cascades capable of giving rise to phrenic motor plasticity. The relative resistance of adenosine-dependent pMF to inflammation suggests that it provides a "backup" system in animals lacking serotonin-dependent pMF due to ongoing inflammation associated with systemic infections and/or neural injury.NEW & NOTEWORTHY This study gives novel insights concerning how a mild systemic inflammation impacts phrenic motor plasticity (pMF), particularly adenosine-dependent pMF. We suggest that since this adenosine-dependent pathway is insensitive to systemic inflammation, it represents an alternative or "backup" mechanism of pMF when other mechanisms are suppressed.

Monitoring undergraduate student needs and activities at Experimental Biology: APS pilot survey.

Life science professional societies play important roles for undergraduates in their fields and increasingly offer membership, fellowships, and awards for undergraduate students. However, the overall impacts of society-student interactions have not been well studied. Here, we sought to develop and test a pilot survey of undergraduate students to determine how they got involved in research and in presenting at the Experimental Biology (EB) meeting, what they gained from the scientific and career development sessions at the meeting, and how the American Physiological Society (APS) can best support and engage undergraduate students. This survey was administered in 2014 and 2015 to undergraduate students who submitted physiology abstracts for and attended EB. More than 150 students responded (38% response rate). Respondents were demographically representative of undergraduate students majoring in life sciences in the United States. Most students (72%) became involved in research through a summer research program or college course. They attended a variety of EB sessions, including poster sessions and symposia, and found them useful. Undergraduate students interacted with established researchers at multiple venues. Students recommended that APS provide more research fellowships (25%) and keep in touch with students via both e-mail (46%) and social media (37%). Our results indicate that APS' EB undergraduate activities are valued by students and are effective in helping them have a positive scientific meeting experience. These results also guided the development of a more streamlined survey for use in future years.

Intermittent hypoxia and stem cell implants preserve breathing capacity in a rodent model of amyotrophic lateral sclerosis.

RATIONALE: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES: To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS: SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.

Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation.

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.

Spinal atypical protein kinase C activity is necessary to stabilize inactivity-induced phrenic motor facilitation.

The neural network controlling breathing must establish rhythmic motor output at a level adequate to sustain life. Reduced respiratory neural activity elicits a novel form of plasticity in circuits driving the diaphragm known as inactivity-induced phrenic motor facilitation (iPMF), a rebound increase in phrenic inspiratory output observed once respiratory neural drive is restored. The mechanisms underlying iPMF are unknown. Here, we demonstrate in anesthetized rats that spinal mechanisms give rise to iPMF and that iPMF consists of at least two mechanistically distinct phases: (1) an early, labile phase that requires atypical PKC (PKCζ and/or PKCι/λ) activity to transition to a (2) late, stable phase. Early (but not late) iPMF is associated with increased interactions between PKCζ/ι and the scaffolding protein ZIP (PKCζ-interacting protein)/p62 in spinal regions associated with the phrenic motor pool. Although PKCζ/ι activity is necessary for iPMF, spinal atypical PKC activity is not necessary for phrenic long-term facilitation (pLTF) following acute intermittent hypoxia, an activity-independent form of spinal respiratory plasticity. Thus, while iPMF and pLTF both manifest as prolonged increases in phrenic burst amplitude, they arise from distinct spinal cellular pathways. Our data are consistent with the hypotheses that (1) local mechanisms sense and respond to reduced respiratory-related activity in the phrenic motor pool and (2) inactivity-induced increases in phrenic inspiratory output require local PKCζ/ι activity to stabilize into a long-lasting iPMF. Although the physiological role of iPMF is unknown, we suspect that iPMF represents a compensatory mechanism, assuring adequate motor output in a physiological system in which prolonged inactivity ends life.

Minimally Invasive Murine Laryngoscopy for Close-Up Imaging of Laryngeal Motion during Breathing and Swallowing.

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to impair laryngeal function, which results in difficulty breathing (dyspnea), swallowing impairment (dysphagia), and/or voice impairment (dysphonia). Dysphagia, in particular, can lead to aspiration pneumonia and associated morbidity, recurrent hospitalization, and early mortality. Despite these serious consequences, existing treatments for laryngeal dysfunction are largely aimed at surgical and behavioral interventions that unfortunately do not typically restore normal laryngeal function, thus highlighting the urgent need for innovative solutions. To bridge this gap, we have been developing an experimental endoscopic approach to investigate laryngeal dysfunction in murine (i.e., mouse and rat) models. However, endoscopy in rodents is quite challenging due to their small size relative to current endoscope technology, anatomical differences in the upper airway, and the necessity for anesthesia to optimally access the larynx. Here, we describe a novel transoral laryngoscopy approach that permits close-up, unobstructed video imaging of laryngeal motion in mice and rats. Critical steps in the protocol include precise anesthesia management (to prevent overdosing that abolishes swallowing and/or risks respiratory distress-related mortality) and micromanipulator control of the endoscope (for stable video recording of laryngeal motion by a single researcher for subsequent quantification). Importantly, the protocol can be performed over time in the same animals to study the impact of various pathological conditions specifically on laryngeal function. A novel advantage of this protocol is the ability to visualize airway protection during swallowing, which is not possible in humans due to epiglottic inversion over the laryngeal inlet that obstructs the glottis from view. Rodents therefore provide a unique opportunity to specifically investigate the mechanisms of normal versus pathological laryngeal airway protection for the ultimate purpose of discovering treatments to effectively restore normal laryngeal function.

Nonsteroidal anti-inflammatory drug (ketoprofen) delivery differentially impacts phrenic long-term facilitation in rats with motor neuron death induced by intrapleural CTB-SAP injections.

Intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) selectively eliminates respiratory (e.g., phrenic) motor neurons, and mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Additionally, microglial density increases in the phrenic motor nucleus following CTB-SAP. This CTB-SAP rodent model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons, and the underlying mechanisms that contribute to enhancing or constraining their output at 7 days (d) or 28d post-CTB-SAP injection. 7d CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) through the Gs-pathway (inflammation-resistant in naïve rats), while pLTF is elicited though the Gq-pathway (inflammation-sensitive in naïve rats) in control and 28d CTB-SAP rats. In 7d and 28d male CTB-SAP rats and controls, we evaluated the effect of cyclooxygenase-1/2 enzymes on pLTF by delivery of the nonsteroidal anti-inflammatory drug, ketoprofen (IP), and we hypothesized that pLTF would be unaffected by ketoprofen in 7d CTB-SAP rats, but pLTF would be enhanced in 28d CTB-SAP rats. In anesthetized, paralyzed and ventilated rats, pLTF was surprisingly attenuated in 7d CTB-SAP rats and enhanced in 28d CTB-SAP rats (both p < 0.05) following ketoprofen delivery. Additionally in CTB-SAP rats: 1) microglia were more amoeboid in the phrenic motor nucleus; and 2) cervical spinal inflammatory-associated factor expression (TNF-α, BDNF, and IL-10) was increased vs. controls in the absence of ketoprofen (p < 0.05). Following ketoprofen delivery, TNF-α and IL-10 expression was decreased back to control levels, while BDNF expression was differentially affected over the course of motor neuron death in CTB-SAP rats. This study furthers our understanding of factors (e.g., cyclooxygenase-1/2-induced inflammation) that contribute to enhancing or constraining pLTF and its implications for breathing following respiratory motor neuron death.

Axon and Myelin Sheath Segmentation in Electron Microscopy Images using Meta Learning.

Various neurological diseases affect the morphology of myelinated axons. Quantitative analysis of these structures and changes occurring due to neurodegeneration or neuroregeneration is of great importance for characterization of disease state and treatment response. This paper proposes a robust, meta-learning based pipeline for segmentation of axons and surrounding myelin sheaths in electron microscopy images. This is the first step towards computation of electron microscopy related bio-markers of hypoglossal nerve degeneration/regeneration. This segmentation task is challenging due to large variations in morphology and texture of myelinated axons at different levels of degeneration and very limited availability of annotated data. To overcome these difficulties, the proposed pipeline uses a meta learning-based training strategy and a U-net like encoder decoder deep neural network. Experiments on unseen test data collected at different magnification levels (i.e, trained on 500X and 1200X images, and tested on 250X and 2500X images) showed improved segmentation performance by 5% to 7% compared to a regularly trained, comparable deep learning network.

A Strength Endurance Exercise Paradigm Mitigates Deficits in Hypoglossal-Tongue Axis Function, Strength, and Structure in a Rodent Model of Hypoglossal Motor Neuron Degeneration.

The tongue plays a crucial role in the swallowing process, and impairment can lead to dysphagia, particularly in motor neuron diseases (MNDs) resulting in hypoglossal-tongue axis degeneration (e.g., amyotrophic lateral sclerosis and progressive bulbar palsy). This study utilized our previously established inducible rodent model of dysphagia due to targeted degeneration of the hypoglossal-tongue axis. This model was created by injecting cholera toxin B conjugated to saporin (CTB-SAP) into the genioglossus muscle of the tongue base for retrograde transport to the hypoglossal (XII) nucleus via the hypoglossal nerve, which provides the sole motor control of the tongue. Our goal was to investigate the effect of high-repetition/low-resistance tongue exercise on tongue function, strength, and structure in four groups of male rats: (1) control + sham exercise (n = 13); (2) control + exercise (n = 10); (3) CTB-SAP + sham exercise (n = 13); and (4) CTB-SAP + exercise (n = 12). For each group, a custom spout with adjustable lick force requirement for fluid access was placed in the home cage overnight on days 4 and 6 post-tongue injection. For the two sham exercise groups, the lick force requirement was negligible. For the two exercise groups, the lick force requirement was set to ∼40% greater than the maximum voluntary lick force for individual rats. Following exercise exposure, we evaluated the effect on hypoglossal-tongue axis function (via videofluoroscopy), strength (via force-lickometer), and structure [via Magnetic Resonance Imaging (MRI) of the brainstem and tongue in a subset of rats]. Results showed that sham-exercised CTB-SAP rats had significant deficits in lick rate, swallow timing, and lick force. In exercised CTB-SAP rats, lick rate and lick force were preserved; however, swallow timing deficits persisted. MRI revealed corresponding degenerative changes in the hypoglossal-tongue axis that were mitigated by tongue exercise. These collective findings suggest that high-repetition/low-resistance tongue exercise in our model is a safe and effective treatment to prevent/diminish signs of hypoglossal-tongue axis degeneration. The next step is to leverage our rat model to optimize exercise dosing parameters and investigate corresponding treatment mechanisms of action for future translation to MND clinical trials.

Phrenic motor neuron survival below cervical spinal cord hemisection.

Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.